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Nucleic Acids Res. 2017 Jul 27;45(13):7581-7592. doi: 10.1093/nar/gkx507.

5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.

Author information

1
RNA Therapeutics Institute, University of Massachusetts Medical School, 01605 Worcester, MA, USA.
2
Program in Molecular Medicine, University of Massachusetts Medical School, 01605 Worcester, MA, USA.
3
Department of Medicine, University of Massachusetts Medical School, 01605 Worcester, MA, USA.

Abstract

5΄-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5΄-vinylphosphonate confers novel properties to siRNAs. Specifically, 5΄-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5΄-to-3΄ exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5΄-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5΄-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5΄-phosphate is removed within 2 hours. The 5΄-phosphate mimic 5΄-(E)-vinylphosphonate stabilizes the 5΄ end of the guide strand by protecting it from phosphatases and 5΄-to-3΄ exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5΄-(E)-vinylphosphonate stabilizes 5΄ phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.

PMID:
28591791
PMCID:
PMC5570069
DOI:
10.1093/nar/gkx507
[Indexed for MEDLINE]
Free PMC Article

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